Days before he was gunned down while riding his bicycle on Sunday, lawyer Rodrigo Rosenberg recorded a video accusing Colom of ordering his death. He named Colom's wife and his personal secretary as co-conspirators.
Some 1,500 people marched to the Constitution Plaza Wednesday demanding Colom's resignation.
"We cannot allow these people to continue doing what they want with our country," said protester Ricardo Flores Asturias, brother of former vice president Luis Flores Asturias (1996-2000).
A separate group of some 600 Colom supporters, mostly residents of working class neighborhoods, marched to within 150 meters (yards) and jeered at the protesters.
"This is a government of the poor!" the crowd chanted. "Stop being corrupt!"
Colom is a center-left social democrat whose social programs and tax programs have rattled the country's conservative elite. He took office in January 2008.
Some 250 of Guatemala's 331 mayors offered Colom their support on Tuesday.
"If at this moment you are hearing or watching this message, it is because Alvaro Colom assassinated me," the slain Rosenberg says in the 18-minute tape, which has been widely viewed in Guatemala.
Rosenberg, 48, who received graduate training at Cambridge and Harvard Universities, represented Khalil Musa, a leading Guatemalan industrialist killed in a hail of bullets along with his adult daughter on April 15.
Rosenberg's murder is "part of a chain of events over the last months" linked to organized crime, said Organization of American States Secretary General Jose Miguel Insulza.
The Washington-based OAS gave Colom its full support on Wednesday, approving a resolution supporting his administration "in its obligation to preserve the institutions of democracy and the rule of law."
Insulza said he will visit Guatemala next week to meet people involved in the case and help support the investigation.
Foreign Minister Haroldo Rodas at the OAS earlier condemned Rosenberg's killing and vehemently rejected charges of Colom's involvement.
In Guatemala, Attorney General Amilcar Velasquez Zarate told AFP that he was handing the Rosenberg case over to the UN-supported International Commission against Impunity in Guatemala (CICIG) to guarantee its independence.
"We have no interest in hiding anything, or anyone," Velasquez said. "We want to get to the bottom of this investigation"
The CICIG chief, Spaniard Carlos Castresana, said he personally asked Colom to not intervene in the investigation.
Four Nobel Peace winners meeting in Guatemala for a separate event expressed their concern over the case.
"We are in an unprecedented crisis that makes clear the vulnerabilities of the institutional system," said Guatemalan indigenous leader Rigoberta Menchu, the 1992 Nobel winner.
She was joined by Iranian 2003 winner Shirin Ebadi, 1997 US winner Jody Williams, and 1976 British winner Mairead Corrigan-Maguire in expressing concern and support for the country's young democracy.
Some 98 percent of criminal cases in Guatemala go unsolved, according to CICIG figures.
US ambassador Stephen McFarland also announced that an FBI agent had arrived to help the CICIG investigation, following Colom's request for foreign help.
Like many Central American countries, Guatemala is used as both a bridge and a storage spot by illegal drug cartels moving cocaine from South America to markets in the United States and Europe.
Guatemala is also still recovering from the effects of the 1960-1996 civil war, in which leftist insurgents battled the country's armed forces, which often responded with scorched earth tactics. Some 200,000 people were killed during that period, according to human rights groups.